Project Summary/Abstract Although double stranded breaks (DSBs) are normally generated in response to stimuli that cause DNA damage, they are also continuously generated in immature thymocytes (DN3 stage) undergoing V(D)J recombination. While coding ends are processed and re-joined quickly by the non-homologous end-joining (NHEJ) pathway at the G1/S cell cycle checkpoint, we have shown that signal ends (DSBs from the excised DNA fragment) remain in post-G1 DN3 thymocytes. These signal ends can randomly integrate in the genome causing genomic instability that may lead to T cell malignancies. We have shown that p38 MAP kinase (MAPK) induces a G2/M cell cycle checkpoint in DN3 thymocytes. We believe that this G2/M cell cycle checkpoint is triggered by the presence of signal ends and it is essential to repair these DSBs and prevent their integration into the genome, thereby preventing genomic instability. Our recent studies indicate that activation of p38 MAPK in response to DSBs causes a preferential nuclear localization of this kinase. We have also shown that p38 MAPK activates the [unreadable]-catenin /survival pathway by phosporylation of GSK3[unreadable]. We propose that the presence of signal ends in DN3 thymocytes leads to activation and nuclear accumulation of p38 MAPK, and that p38 MAPK pathway plays a critical role in facilitating signal ends repair and preventing their random integration into the genome by: 1) inducing a G2/M cell cycle checkpoint for signal ends to be repaired prior to cell division, and 2) inducing survival of DN3 thymocytes while undergoing the G2/M cell cycle checkpoint. We will investigate whether: 1) activation of p38 MAPK by signal ends promotes nuclear accumulation of p38 MAPK through its association with NBS1 to induce a G2/M cell cycle checkpoint in DN3 thymocytes (Aim 1), p38 MAPK promotes survival in DN3 thymocytes while undergoing the G2/M cell cycle checkpoint through phosphorylation and inactivation of GSK3[unreadable] (Aim 2), 3) activation of p38 MAPK and establishment of a G2/M cell cycle checkpoint is essential for repairing signal ends and preventing integration of signal joints into the genome (Aim 3).